Genome-wide identification and expression analysis of the metacaspase gene family in Hevea brasiliensis

Plant Physiology and Biochemistry 105 (2016) 90e101

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Research article

Genome-wide identification and expression analysis of the metacaspase gene family in Hevea brasiliensis Hui Liu a, Zhi Deng a, Jiangshu Chen b, Sen Wang c, Lili Hao c, Dejun Li a, * a

Key Laboratory of Biology and Genetic Resources of Rubber Tree, Ministry of Agriculture, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou 571737, China b College of Agriculture, Hainan University, Haikou 570228, China c CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 January 2016 Received in revised form 5 April 2016 Accepted 5 April 2016 Available online 7 April 2016

Metacaspases, a family of cysteine proteases, have been suggested to play important roles in programmed cell death (PCD) during plant development and stress responses. To date, no systematic characterization of this gene family has been reported in rubber tree (Hevea brasiliensis). In the present study, nine metacaspase genes, designated as HbMC1 to HbMC9, were identified from whole-genome sequence of rubber tree. Multiple sequence alignment and phylogenetic analyses suggested that these genes were divided into two types: type I (HbMC1eHBMC7) and type II (HbMC8 and HbMC9). Gene structure analysis demonstrated that type I and type II HbMCs separately contained four and two introns, indicating the conserved exoneintron organization of HbMCs. Quantitative real-time PCR analysis revealed that HbMCs showed distinct expression patterns in different tissues, suggesting the functional diversity of HbMCs in various tissues during development. Most of the HbMCs were regulated by drought, cold, and salt stress, implying their possible functions in regulating abiotic stress-induced cell death. Of the nine HbMCs, HbMC1, HbMC2, HbMC5, and HbMC8 displayed a significantly higher relative transcript accumulation in barks of tapping panel dryness (TPD) trees compared with healthy trees. In addition, the four genes were up-regulated by ethephon (ET) and methyl jasmonate (MeJA), indicating their potential involvement in TPD resulting from ET- or JA-induced PCD. In summary, this work provides valuable information for further functional characterization of HbMC genes in rubber tree. © 2016 Elsevier Masson SAS. All rights reserved.

Keywords: Metacaspase Hevea brasiliensis Tapping panel dryness Programmed cell death Abiotic stress

1. Introduction Programmed cell death (PCD) is a conserved and genetically controlled cell death process. In plants, PCD includes two broad categories, developmentally regulated PCD and environmentally induced PCD (Gunawardena, 2008). Developmentally regulated PCD covers a wild range of tissues and organs, such as leaf, xylem, embryo, etc. (Bollhoner et al., 2013; Huang et al., 2014; Wertman

Abbreviations: EST, expressed sequence tag; ET, ethephon; LSD, lesion-simulating disease; MC, metacaspase; MeJA, methyl jasmonate; ORF, open reading frame; PCD, programmed cell death; qRT-PCR, quantitative real-time PCR; TPD, tapping panel dryness; TSA, transcriptome shotgun assembly. * Corresponding author. Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Baodao Xincun, Danzhou, Hainan 571737, China. E-mail addresses: [email protected] (H. Liu), [email protected] (Z. Deng), [email protected] (J. Chen), [email protected] (S. Wang), [email protected] (L. Hao), [email protected] (D. Li). http://dx.doi.org/10.1016/j.plaphy.2016.04.011 0981-9428/© 2016 Elsevier Masson SAS. All rights reserved.

et al., 2012), and it is initiated by the internal factors and occurs at a predictable time and location (Gunawardena, 2008). In contrast, environmentally induced PCD is triggered by external biotic or abiotic signals, such as pathogen, heat shock, and water stress (Duan et al., 2010; Kim et al., 2013; Li et al., 2012; OlveraCarrillo et al., 2015). PCD is essential for plant development and survival against pathogen invasion and environmental stresses. Despite the importance of PCD in plants, the molecular mechanisms involved in this process are largely unclear. However, in animals, the molecular mechanisms of PCD have been well elucidated by studying the model system Caenorhabditis elegans (Lord and Gunawardena, 2012). In animal cells, caspases (cysteine aspartic-specific proteases) play central role in signaling and executing PCD (Grutter, 2000). However, no orthologous caspases have been identified in plants. The only plant gene family closely resembling caspases is the metacaspase family (Uren et al., 2000). Although metacaspases have similar morphology and secondary structure as caspases, they

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cannot be defined as caspases, since metacaspases do not have aspartate-specific proteolytic activity (Tsiatsiani et al., 2011). Plant metacaspases were divided into type I and type II based on their sequence and structure similarity (Tsiatsiani et al., 2011; Fagundes et al., 2015). Both type I and II metacaspases have a putative conserved caspase-like catalytic domain composed of p10 and p20 subunits, which contain conserved catalytic histidine/cysteine (His/ Cys) dyad (Fagundes et al., 2015; Vercammen et al., 2007). The catalytic histidine lies in the (H/Y)(Y/F)SGHG sequence and the catalytic cysteine in the active-site, pentapeptide D(A/S)C(H/Y)S sequence (Fagundes et al., 2015; Zhang et al., 2013). Besides, type I metacaspases could present or not present a prodomain rich in proline, include a zinc finger motif in the N-terminus region. Whereas type II metacaspases lack the prodomain and the zinc finger motif, but harbor a longer linker region than that found in type I metacaspases, which connects the p10 and p20 subunits (Fagundes et al., 2015). Metacaspases play important roles in plant PCD (Fagundes et al., 2015; Lam and Zhang, 2012). Several metacaspase genes have been demonstrated to be essential for different types of PCD in plants. In Arabidopsis, there are three type I (AtMC1eAtMC3, also known as AtMCP1a-AtMCP1c) and six type II (AtMC4eAtMC9, also known as AtMCP2aeAtMCP2f) metacaspase genes (Tsiatsiani et al., 2011). Among the type I metacaspases, AtMC1 and AtMC2 antagonistically control hypersensitive response-associated cell death in Arabidopsis. AtMC1 is a positive regulator of cell death, whereas AtMC2 negatively regulates cell death (Coll et al., 2010). Among the type II metacaspases, AtMC4 plays a positive regulatory role in biotic and abiotic stress-induced PCD (Watanabe and Lam, 2011), and AtMC8 is required for cell death triggered by UVC and H2O2 (He et al., 2008). Additionally, AtMC9 is essential for efficient progression of autolysis during vessel cell death (Bollhoner et al., 2013; Tsiatsiani et al., 2013). In wheat, the metacaspase gene TaMCA4 functions in PCD induced by the fungal pathogen Puccinia striiformis f. sp. tritici (Wang et al., 2012). Moreover, the pepper metacaspase gene Camc9 plays a role as a positive regulator of pathogen-induced cell death via the regulation of reactive oxygen species production and defence-related gene expression in plants (Kim et al., 2013). In Norway spruce (Picea abies), the metacaspase gene mcII-Pa is required for both progression of vacuolar cell death and suppression of necrosis (Minina et al., 2013). These results indicate that metacaspases are essential for cell death regulation in plants. The metacaspase gene family has been systematically investigated by genome-wide scans in Viridiplantae (Fagundes et al., 2015), Arabidopsis (Kwon and Hwang, 2013; Tsiatsiani et al., 2011), grape (Zhang et al., 2013), and rice (Huang et al., 2015; Wang and Zhang, 2014). In addition, several studies on metacaspases have been reported in maize (Ahmad et al., 2012), tomato (Hoeberichts et al., 2003), pepper (Kim et al., 2013), and wheat (Wang et al., 2012). However, to date, no metacaspase gene has been reported in rubber tree (Hevea brasiliensis). Rubber tree is a perennial plant in the Euphorbiaceae family and is the sole commercial source of natural rubber because of its high production and rubber quality. Tapping panel dryness (TPD) ac
belin1 et al., counts for 10e40% annual rubber production losses (Ge 2015), therefore it is one of the most serious threats to natural rubber production. The TPD syndrome is characterized by the partial or complete cessation of latex flow upon tapping (Venkatachalam et al., 2007). Previous studies suggested that PCD in bark cells possibly play a role in TPD occurrence (Chen et al., 2003; Li et al., 2010; Putranto et al., 2015; Venkatachalam et al., 2007). Metacaspases, as important regulators of PCD, may be associated with TPD. The completion of rubber tree genome sequence has made it possible to identify and characterize the metacaspase family genes at a genome-wide level. In the present

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study, we identified nine rubber tree metacaspase genes (HbMC1eHbMC9) and analyzed their gene structure, phylogenetic relationship, expression profiles in various tissues, and response to different types of abiotic stress and hormone treatments. Our results lay the foundation for future functional characterization of HbMC genes in rubber tree. 2. Materials and methods 2.1. Plant materials and treatments Rubber tree clone, Reyan 7-33-97, was cultivated under normal field conditions at the experimental farm of Chinese Academy of Tropical Agricultural Sciences in Danzhou, Hainan, China. The fresh tissues or organs including leaf, stem tip, latex, bark, female flower, and male flower were sampled from 20-year-old rubber trees during spring bloom period. Roots were collected from one-yearold tissue culture seedlings of Reyan 7-33-97. Latex and barks were collected from healthy and TPD rubber trees selected according to Li et al. (2010). Three biological replicates were sampled for each tissue, and each replicate was equally harvested from five trees. Samples were immediately frozen in liquid nitrogen and then stored at
80  C for RNA isolation. The tissue culture seedlings of Reyan 7-33-97 were used for cold, drought, and salt stress treatments. Cold stress treatment was performed by transferring seedlings to a growth chamber at 8  C. For drought and salt stress treatments, the seedlings were washed thoroughly with tap water to eliminate substrates, and then transferred into solutions supplemented with 20% (W/V) PEG-6000 (polyethylene glycol 6000) or 1 M NaCl, respectively. Each treatment had three replicates, and each replicate contained three seedlings. Leaf samples were collected at 0, 3, 24, and 48 h after treatments, and then immediately frozen into liquid nitrogen and stored at
80  C for RNA extraction. The seven-year-old virgin trees were used for ethephon (ET), methyl jasmonate (MeJA), and wounding treatments. ET and MeJA treatments were carried out according to the methods of Hao and Wu (2000). Latex was harvested at 0, 4, 8, 24, and 48 h after treatments. The wounding treatment was performed as described by Tang et al. (2010). Latex was harvested at 0, 6, 24, and 48 h after treatment. Each treatment had three replicates, and each replicate contained three trees. The first few drops of latex containing the debris were discarded, and then the latex from the treated and control rubber tree was allowed to drop directly into liquid nitrogen in an ice kettle for total RNA extraction. 2.2. RNA isolation and first-strand cDNA synthesis Total RNA was isolated from the collected samples according to Xu's method (Xu et al., 2010), and then treated with RQ1 RNase-free DNase (Promega, USA) to remove genomic DNA contamination. The quality and quantity of the extracted RNA were checked by agarose gel electrophoresis and measured by a spectrophotometer (Thermo Scientific NanoDrop 2000, USA). First strand cDNA was synthesized with RevertAid™ First Strand cDNA Synthesis Kit (Thermo Scientific, USA) according to the manufacturer's instruction. 2.3. Identification and isolation of metacaspase genes in Hevea brasiliensis The nine full-length cDNA sequences of Arabidopsis thaliana metacaspase genes (AtMC1eAtMC9) were obtained from TAIR (http://www.arabidopsis.org/) as reported in previous study (Tsiatsiani et al., 2011). The cDNA sequences of these genes were used as queries to search against the Transcriptome Shotgun

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Assembly (TSA) and Expressed Sequence Tags (EST) of Hevea brasiliensis at NCBI (http://www.ncbi.nlm.nih.gov/), and the rubber tree genome sequenced by Centre for Chemical Biology, Universiti Sains Malaysia (http://bioinfo.ccbusm.edu.my/cgi-bin/gb2/ gbrowse/Rubber/) (Rahman et al., 2013) or by Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences (unpublished data). Redundant sequences were removed after similarity comparison. The open reading frames (ORFs) of candidate mRNA or genome DNA sequences were determined by NCBI ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and Softberry (http://linux1.softberry.com/). All identified HbMCs were further validated by conserved domain searching using CDD (http://www. ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and InterProScan (http://www.ebi.ac.uk/interpro/scan.html) to confirm the presence of the caspase-like domain. Based on the predicted sequences, gene-specific primers used to amplify the corresponding full length coding cDNA sequences of HbMCs were designed by Primer3.0 (http://primer3.ut.ee/). The primer pairs for all HbMC genes are listed in Table S1. RT-PCR was performed using Pyrobest™ DNA polymerase (TaKaRa, Japan) with the mixture of cDNA from various tissues as template. The PCR products were cloned into the pMD18-T Vector (TaKaRa, Japan) and then transformed into E.coli competent cells DH5a. Finally, the products were sequenced after screening identification of bacterial colonies by PCR. At least three clones per gene were randomly picked and sequenced. The cDNA sequences of HbMCs were determined using alignment analysis with their corresponding sequences obtained from bioinformatic analysis. 2.4. Protein properties and gene structure analysis of HbMCs The theoretical molecular weight (Mw) and isoelectric point (pI) of HbMC proteins were predicted by the ExPASy's Compute pI/Mw tool (http://web.expasy.org/compute_pi/). The genomic DNA sequences of HbMCs were obtained by the BLASTN search of the rubber tree genome database described above using the cDNA sequences as queries. Exon-intron structures of HbMCs were identified with coding sequence alignments to corresponding genomic sequences using FGENESH-C (http://linux1.softberry.com/berry. phtml?topic¼fgenes_c&group¼programs&subgroup¼gfs). 2.5. Multiple sequence alignments and phylogenetic analysis Amino acid sequence identity of HbMC proteins was calculated by using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/ ). The metacaspase protein and cDNA sequences of Arabidopsis, rice, and grape were obtained from TAIR (http://www.arabidopsis. org/), MSU Rice Genome Annotation Project, and GenBank according to previous studies (Tsiatsiani et al., 2011; Wang and Zhang, 2014; Zhang et al., 2013). Multiple alignments of HbMC and AtMC proteins were carried out using BioEdit software. The Bayesian phylogenetic tree was constructed using BEAST v.1.8.3 (Drummond and Rambaut, 2007), which was performed as described by Cabreira et al. (2015). Mus musculus caspase gene Casp1 (NM_009807.2) was used as an outgroup to root the tree. 2.6. Quantitative real-time PCR (qRT-PCR) analysis qRT-PCR was performed with SYBR® Premix Ex Taq™ (TaKaRa, Japan) using the CFX96™ Real-Time System (Bio-Rad, USA), according to the suppliers' manuals. The thermal cycle was as follows: 95  C for 1 min, followed by 40 cycles of 95  C for 5 s and 60  C for 20 s. Melting curve was routinely performed after 40 cycles to verify primer specificity. Three technical replicates were run for each biological sample. The 18S rRNA gene (GenBank accession No.:

AB268099) was used as the internal control (Tang et al., 2010). All primers were designed by Primer3 (http://frodo.wi.mit.edu/ primer3). The primer sequences and their efficiencies are given in Table S2. 2.7. Statistical analysis Data analysis and graphical visualization was carried out using SigmaPlot 12 software. The relative expression level was calculated using the 2
DDCT method, in which CT indicates cycle threshold (Livak and Schmittgen, 2001). Data were expressed as the mean ± SD (standard deviation) of three biological replicates. Statistical analysis was performed by Tukey's test or t-test. 3. Results 3.1. Identification and characterization of HbMC genes in rubber tree To identify the members of metacaspase gene family in rubber tree, the previously reported Arabidopsis metacaspase full-length cDNAs were used as the query sequences to search against the EST, TSA, and genome database of rubber tree with BLASTN program. The candidates were then examined by CDD and InterProScan to confirm the presence of the caspase-like domain. After removing the redundant sequences, a total of nine non-redundant metacaspase genes (designated as HbMC1eHbMC9) with complete ORFs were identified in rubber tree (Table 1). To confirm the putative HbMCs, these complete ORF sequences of HbMCs were isolated through PCR-based approaches and sequenced. The accurate cDNA sequences of HbMCs have been deposited in GenBank with accession numbers listed in Table 1. The ORF length of HbMCs ranged from 978 bp (HbMC9) to 1254 bp (HbMC8), encoding polypeptides ranging from 325 amino acids to 417 amino acids. The corresponding molecular weights varied from 35.52 to 45.95 kDa and the predicted isoelectric points varied from 5.26 (HbMC9) to 8.60 (HbMC5) (Table 1). Pairwise sequence comparisons were carried out to examine the degrees of sequence identities between HbMC proteins. The results are summarized in Table S3. The identities between two HbMCs ranged from 25.20% to 76.45%. The average sequence identity between two HbMCs was 40.34%. The largest identity was observed between HbMC1 and HbMC2 (76.45%). HbMC9 showed the least identities with HbMC4 and HbMC5 (25.20%). 3.2. Analysis of conserved domains and structural features of HbMCs The conserved domain analysis indicated that all of the nine HbMC proteins contained caspase-like domain (InterPro accession No.: IPR029030), suggesting that they belonged to metacaspase gene family (Fig. S1). In addition, HbMC1eHbMC3 possessed a LSD1 (lesion-simulating disease-1)-type zinc finger domain (InterPro accession No.: IPR005735) with the consensus sequence as described by Cabreira et al. (2013), indicating that they belonged to type I with zinc finger domain metacaspases as defined by Fagundes et al. (2015). Sequence alignment of HbMCs with AtMCs revealed the conserved motifs and structural features among metacaspases (Fig. 1). All HbMCs and AtMCs have the conserved caspase-like domain composed of p20 subunit, a linker region, and p10 subunit, containing a caspase-specific catalytic dyad of His/Cys (Uren et al., 2000). The sequence context of the catalytic His and Cys residues separately are (H/Y)(Y/F)SGHG and D(A/S)C(H/Y/N)S, which is agreed with the previously reported metacaspase catalytic

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Table 1 Characteristics of metacaspase family genes in Hevea brasiliensis. Gene name

HbMC1 HbMC2 HbMC3 HbMC4 HbMC5 HbMC6 HbMC7 HbMC8 HbMC9

GenBank accession no.

KU188281 KU188282 KU188283 KU188284 KU188285 KU188286 KU188287 KU188288 KU188289

ORF length (bp)

1104 1086 1164 1164 1065 1086 1026 1254 978

Protein

Metacaspase type

Length (aa)

Mw (kDa)

pI

367 361 387 387 354 363 341 417 325

40.24 39.55 42.46 43.58 39.92 40.82 38.47 45.95 35.52

6.22 6.59 5.90 6.89 8.60 8.24 6.56 5.01 5.26

I I I I I I I II II

ORF, open reading frame; bp, base pair; aa, amino acids; Mw, molecular weight; pI, isoelectric point.

site sequences (Fagundes et al., 2015). HbMC1eHbMC7 showed structural similarity to AtMC1eAtMC3 (Fig. 1). Their proteins contain a proline- or glutamine-rich N-terminal prodomain and a shorter linker region between the p20 and p10 subunits, which was in accordance with the characteristics of type I metacaspases. Thus, they belonged to type I HbMCs. The protein structural of HbMC8 and HbMC9 were similar to AtMC4eAtMC9 (Fig. 1). They lack the N-terminal prodomain but harbor a longer linker region between the p20 and p10 subunits, which corresponds to the characteristics of type II metacaspases. Thus, they belonged to type II HbMCs. 3.3. Gene structures and phylogenetic analysis of HbMCs In order to gain further insight into the structural diversity of HbMC genes, we performed an exon/intron analysis by aligning ORF sequences of HbMCs with their corresponding genomic sequences (Fig. 2). According to their predicted structures, the nine HbMCs could be divided into two groups. The first group HbMCs consists of five exons interrupted by four introns, including all the type I HbMCs (HbMC1eHbMC7). The other group included all the type II HbMCs (HbMC8 and HbMC9), which contain two exons interrupted by one intron. These results suggested that HbMCs within the same type shared conserved exoneintron structures. To obtain insight into the evolutionary history and phylogenetic relationships of the HbMC genes, a Bayesian phylogenetic tree was constructed using BEAST software on the basis of multiple sequence alignment of metacaspase family genes from Arabidopsis, rubber tree, rice, and grape (Fig. 3). According to the phylogenetic tree, the 32 metacaspase genes were divided into two clades (I and II). Clade I consisted of 18 type I metacaspase genes, and it was further categorized into three subclades: A, B, and C, with each subclade containing seven, seven, and four members, respectively. Clade II contained 14 type II metacaspase genes, and it was also divided into three subclades: D, E, and F, with each subclade containing four, three, and seven members, respectively. The nine HbMCs were distributed in four subclades: A (HbMC1 and HbMC2), B (HbMC3eHbMC7), D (HbMC9), and F (HbMC8). Subclades C and E were rice-specific subclades. 3.4. Expression profiles of HbMC genes in different tissues of rubber tree To investigate the tissue specificity of HbMCs expression, the expression patterns of HbMCs in various tissues (including roots, leaves, stem tips, barks, latex, male flowers, and female flowers) of rubber tree were analyzed using qRT-PCR. As shown in Fig. 4, HbMCs displayed different tissue expression patterns. HbMC1, HbMC2, HbMC5, and HbMC8 were constitutively expressed in all

tissues tested. Neither HbMC3 nor HbMC9 expression was detected in barks, while HbMC6 and HbMC7 transcripts were not detected in both barks and latex. HbMC4 showed no expression in latex and a significantly lower expression in barks. In contrast, some genes were highly expressed in specific tissues. For example, HbMC8 had a significantly higher expression in latex than other tissues. HbMC5 and HbMC6 had a significantly higher expression in leaves than other tissues. In addition, HbMC1, HbMC7 and HbMC9 were relatively highly expressed in roots and stem tips (Fig. 4). The HbMCs highly expressing in specific tissues may play specific roles in the corresponding tissues. Systematic analyses of TPD-related genes suggested that PCD
belin1 et al., might play important roles in rubber tree TPD (Ge 2015; Li et al., 2010; Venkatachalam et al., 2007). Metacaspases, as key regulators of PCD, possibly play critical roles in TPD occurrence. Therefore, we comparatively analyzed HbMCs expressions between healthy and TPD rubber trees. Among the nine HbMCs, HbMC3, HbMC4, HbMC6, HbMC7, and HbMC9 showed no transcript or no significant changes in latex and barks of healthy and TPD rubber trees (data no shown), indicating that they may not be involved in TPD. In contrast, HbMC1, HbMC5, and HbMC8 showed significantly higher expression in both latex and barks of TPD trees than that of healthy trees (Fig. 5). Additionally, the relative expression level of HbMC2 was significantly higher in barks of TPD trees than that of healthy trees (Fig. 5). These results suggested that HbMC1, HbMC2, HbMC5, and HbMC8 may be involved in TPD occurrence.

3.5. Expression patterns of HbMCs in response to abiotic stress Metacaspases are important regulators of PCD during stress responses in plants (Fagundes et al., 2015; Huang et al., 2015). To explore the possible involvement of HbMCs in response to abiotic stress, the expression patterns of HbMCs under cold, drought, and salt stresses were analyzed by qRT-PCR. As shown in Fig. 6, the expressions of six HbMC genes were regulated by cold stress, and the rest three genes, HbMC2, HbMC5, and HbMC6, showed no obvious transcriptional changes under cold stress. Among the six cold-regulated HbMCs, HbMC1, HbMC7, and HbMC9 were significantly up-regulated at all the treated time-points. HbMC8 showed significant up-regulation only at 24 h after treatment. Only two HbMC genes, HbMC3 and HbMC4, exhibited down-regulated expression in response to cold stress. HbMC4 were dramatically suppressed at 3 and 24 h, while HbMC3 showed significant downregulation at 24 and 48 h (Fig. 6). Under PEG-induced drought stress, only HbMC5 expression was not significantly changed (Fig. 7). Of the eight drought-responsive HbMC genes, four genes (HbMC1, HbMC2, HbMC8, and HbMC9) showed significant up-regulation at least one time-point after

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Fig. 1. Multi-sequence alignment of metacaspase proteins from rubber tree (Hb) and Arabidopsis (At). The rectangle indicate LSD1-type zinc finger domain. The solid line, double solid line, dotted lines, and double dotted lines indicate the Pro/Gln-Rich N-terminal Prodomain, p20 subunit, linker, and p10 subunit, respectively.

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Fig. 2. Exoneintron structures of HbMC genes. The first exons are represented by red boxes. Internal exons are represented by grey boxes and the last exons are represented by blue boxes. Scales show the length of each gene's exons and introns in bp. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

treatment (Fig. 7). Among them, HbMC1 and HbMC2 displayed similar expression patterns upon drought stress. Both of them were rapidly and strongly induced by drought stress, reached the maximum level at 3 h after treatment (6.4- and 2.6-fold of the control for HbMC1 and HbMC2, respectively), and then declined. In contrast, HbMC9 transcripts exhibited continuous increases after drought stress, and resulting in 4.6-fold increase at 48 h after treatment. HbMC8 showed significant up-regulation at 24 h, only increased 1.5-fold over that at 0 h. A total of four HbMC genes were down-regulated by drought stress (Fig. 7). HbMC6 showed significant down-regulation at all the treated time-points. Both HbMC4 and HbMC7 exhibited down-regulated expression at 24 and 48 h after treatment. While HbMC3 displayed significant repressed expression only at 24 h after treatment. Under salt stress, HbMC2, HbMC4, and HbMC7 showed similar expression patterns (Fig. 8). Their transcripts were rapidly upregulated in response to salt stress, peaked at 3 h of treatment, and then declined. After 48 h of treatment, the transcript abundance of HbMC2 recovered to normal level, whereas the expression levels of HbMC4 and HbMC7 declined to less than 0.2-fold of the control. Additionally, the expression of HbMC3 and HbMC6 also showed significant down-regulation at relatively later stages (24 and/or 48 h after treatment). By contrast, HbMC1 exhibited upregulated expression at 24 h after treatment (Fig. 8). The remaining three HbMC genes (HbMC5, HbMC8, and HbMC9) displayed no significant expression change after salt stress treatment (Fig. 8). 3.6. Expression patterns of HbMCs in response to wounding Farmers harvest latex by regularly tapping the trunk bark of the rubber tree. Tapping is mechanical wounding, and latex leakage is a wounding response of rubber tree. To investigate whether mechanical wounding had any effect on HbMCs, the expression patterns of HbMCs in latex after wounding were analyzed (Fig. 9). No

HbMC4, HbMC6, and HbMC7 transcripts were detected in latex after wounding treatment, which was in accordance with the tissue expression patterns of HbMC4, HbMC6, and HbMC7. Additionally, HbMC2 and HbMC9 showed no obvious transcription level change (data no shown), suggesting they may not be involved in rubber tree wounding response. The expressions of four HbMCs in latex were induced or repressed after wounding treatment (Fig. 9). Of these, HbMC1 expression was significantly up-regulated in response to wounding across all time-points, whereas HbMC3 exhibited significantly down-regulated expression in latex in response to wounding across all time-points. Besides, HbMC5 and HbMC8 were significantly up-regulated by wounding treatment at different stages. HbMC5 was significantly induced, leading to approximately 3.8-fold increase at 24 h after treatment, while HbMC8 was slightly increased by 1.6-fold at 48 h after treatment, as compared with that in control plants without wounding treatment. 3.7. Expression patterns of HbMCs in response to ET and MeJA Jasmonic acid and ethylene regulate cell death under stress conditions and during development (Lam, 2004). Moreover, ethylene and jasmonic acid play critical roles in regulating latex production in rubber tree (Hao and Wu, 2000; Zhu and Zhang, 2009). Given the major roles of ethylene and jasmonic acid in regulating latex biosynthesis and cell death, we further analyzed the expression of HbMCs in latex in response to exogenous ET and MeJA treatments. HbMC4, HbMC6, and HbMC7 still showed no expression in latex after ET and MeJA treatments. Additionally, there was no significant change in the expressions of HbMC3 and HbMC9 (data not shown). Only four HbMCs were regulated by ET and JA treatments (Fig. 10). After ET treatment, HbMC1 and HbMC5 displayed significantly up-regulated expression across all timepoints, and HbMC2 exhibited significant up-regulation only at 8 h. HbMC8 displayed an irregular expression pattern. It was up-

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Fig. 3. Phylogenetic relationships of the metacaspase family proteins from rubber tree, Arabidopsis, rice and grape. The Bayesian phylogenetic tree was constructed using BEAST v1.8.3. The caspase gene Casp1 (NM_009807.2) from Mus musculus was used as an outgroup to root the tree. The posterior probabilities are given for each node in the tree. The six subclades are indicated with different colors. The metacaspase family genes of Arabidopsis (At), rice (Os), and grape (Vv) were described according to previous studies (Tsiatsiani et al., 2011; Wang and Zhang, 2014; Zhang et al., 2013). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

regulated at 8 h, but was suppressed at 4 and 48 h. Interestingly, all of these four HbMCs showed the highest expression levels at 8 h, 14.5-, 2.3-, 10.6-, and 1.9-fold up-regulation for HbMC1, HbMC2, HbMC5, and HbMC8, respectively. After MeJA treatment, HbMC1, HbMC2, and HbMC5 transcripts increased gradually, peaked at 8 h, and then recovered to normal levels after 24 h. HbMC8 transcripts were also gradually accumulated, but it reached the maximum level at 24 h after treatment, and increased only 1.6-fold over that at 0 h. 4. Discussion Metacaspases are cysteine proteases and widespread presence in Viridiplantae, from algae to vascular plants. The number of metacaspase genes in the genomes of different plant species varies considerably, from one in some of green algae to 20 in Brassica rapa Chiifu-FPsc (Fagundes et al., 2015). However, to our knowledge, the metacaspase gene family from rubber tree has not been characterized in detail. In this study, we carried out genome-scale identification and expression analysis of metacaspase gene family in rubber tree. A total of nine HbMC genes were identified in rubber tree genome, which is equal to the number of metacaspase genes in Arabidopsis (Tsiatsiani et al., 2011). Of the nine AtMCs, three belonged to the type I metacaspases, and the other six were type II metacaspases. However, the rubber tree possessed seven type I metacaspases and only two type II metacaspases (Figs. 1 and 3).

More recently, Fagundes et al. (2015) adopted a comparative genomic approach to identify metacaspase genes in Viridiplantae. They predicted the distribution of type I and II metacaspases in 42 plant species. The total number of type I metacaspases was 259, which is more than twice the number of type II metacaspases, suggesting that most of plant species have greater numbers of type I metacaspases relative to type II. Gene structure analysis revealed that type I HbMCs had five exons interrupted by four introns, and type II HbMCs consisted of two exons and a single intron (Fig. 2). The same exon/intron structures were obtained from the analysis of grape VvMCs (Zhang et al., 2013) and Arabidopsis type II AtMC4 (Watanabe and Lam, 2011). We further analyzed the gene structure of the other AtMCs and found that type I AtMC1eAtMC3 also had the 5 exon/4 intron structures, and type II AtMC5eAtMC8 also had the 2 exon/1 intron structures. However, type II AtMC9 only had one exon. Rice type I OsMCs had 3e5 exons, and type II OsMCs had one or two exons (Wang and Zhang, 2014). These results suggested that type I metacaspases had more exon/intron numbers than type II metacaspases. The type I metacaspases may present or not present a zinc finger domain in the N-terminus region (Fagundes et al., 2015). In the present study, zinc finger domain was found in type I metacaspases HbMC1eHbMC3 but not in HbMC4eHbMC7. It has been hypothesized that the acquisition of the zinc finger domain seems to have

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Fig. 4. Expression patterns of HbMC genes in different tissues of rubber tree. Relative expression levels of HbMC genes were determined by qRT-PCR and normalized by the 18S rRNA gene expression. For each gene, the transcript level in leaf was used to normalize the transcript levels in other tissues. Values are means ± SD of three biological replicates. Different letters indicate significant differences among the different tissues (P < 0.05, Tukey's test). Ro, roots; St, stem tips; Le, leaves; Ba, Barks; La, latex; Mf, male flowers; Ff, female flowers.

Fig. 5. Comparative analysis of expression levels of HbMCs in latex and barks of the healthy and TPD rubber trees. Relative expression levels of HbMC genes were determined by qRT-PCR and normalized by the 18S rRNA gene expression. For each gene, the transcript level in barks of healthy tree was used to normalize the transcript level in other tissues. Values are means ± SD of three biological replicates. Asterisks indicate a significant difference (*, P < 0.05; **, P < 0.01, t-test) between the TPD and healthy rubber trees.

occurred later during the metacaspase gene family evolution (Fagundes et al., 2015). Thus, HbMC4eHbMC7 should be originated in the early stage of Hevea brasiliensis evolution. It has been proved

that the LSD1-type zinc finger domains in Arabidopsis AtMC1 and rice OsMC1 were required for interaction with LSD proteins (Coll et al., 2010; Huang et al., 2015). Whether HbMC1eHbMC3 can interact with HbLSDs and their zinc finger domains are required for the interaction still need to be examined. Comprehensive gene expression analysis of metacaspase family genes revealed that the metacaspases had distinct expression patterns in various tissues of Arabidopsis, rice, and grape (Kwon and Hwang, 2013; Wang and Zhang, 2014; Zhang et al., 2013). Consistent with the aforementioned results, HbMCs also showed apparent differential expression patterns in various tissues. Some HbMCs exhibited high expression levels in specific tissues, implying that they might play specific roles required in these tissue types. For example, HbMC1, HbMC7, and HbMC9 were highly expressed in roots and stem tips, and HbMC6 was abundantly expressed in leaves. Six HbMCs were expressed in latex. Of these, HbMC8, also designated as latex-abundant protein (GenBank: AAD13216.1), showed a relatively higher expression level in latex than in other tissues (Fig. 4). Furthermore, the expression of HbMC8 was regulated by ET and MeJA (Fig. 10). ET and MeJA are key signals for latex production in rubber tree (Hao and Wu, 2000; Zhu and Zhang, 2009). These results suggested that HbMC8 may play an important role in latex biosynthesis in rubber tree. Hevea brasiliensis is a native species of the Amazon Basin of South America, but it is widely planted in Southeast Asia, such as Thailand, Vietnam, southern China, etc. Rubber trees planting in these new areas are often subjected to abiotic stresses like low temperature, drought, and typhoons. Diverse abiotic stresses have been found to induce PCD (Petrov et al., 2015). Previous studies have pointed to metacaspase involvement in abiotic-induced PCD

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Fig. 6. Expression profiles of HbMC genes under cold stress. Leaves of seedlings were sampled at 0, 3, 24 and 48 h after cold (8  C) stress treatment. Relative expression level of each gene was normalized with 18S rRNA gene. Data are means ± SD of three biological replicates. Asterisks indicate a significant difference (*, P < 0.05; **, P < 0.01, t-test) compared with the corresponding control (0 h).

Fig. 7. Expression profiles of HbMC genes under drought stress. Leaves of seedlings were sampled at 0, 3, 24 and 48 h after PEG-induced drought stress treatment. Relative expression level of each gene was normalized with 18S rRNA gene. Data are means ± SD calculated from three biological replicates. Asterisks indicate a significant difference (*, P < 0.05; **, P < 0.01, t-test) compared with the corresponding control (0 h).

in pants (Fagundes et al., 2015). In silico analysis of cis-elements in the promoter sequences of grapevine and rice metacaspase family

genes indicated that all of these metacaspase gene promoters contained cis-elements related to stress responses (Huang et al.,

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Fig. 8. Expression profiles of HbMC genes under salt stress. Leaves of seedlings were sampled at 0, 3, 24 and 48 h after salt (1 M NaCl) stress treatment. Relative expression level of each gene was normalized with 18S rRNA gene. Data are means ± SD of three biological replicates. Asterisks indicate a significant difference (*, P < 0.05; **, P < 0.01, t-test) compared with the corresponding control (0 h).

Fig. 9. Expression profiles of HbMC genes in latex of rubber tree responding to wounding. Latex was sampled at 0, 6, 24 and 48 h after wounding treatment. Relative expression level of each gene was normalized with 18S rRNA gene. Data are means ± SD of three biological replicates. Asterisks indicate a significant difference (*, P < 0.05; **, P < 0.01, t-test) compared with the corresponding control (0 h).

Fig. 10. Expression profiles of HbMC genes in latex of rubber tree responding to ET and MeJA treatments. Latex was sampled at 0, 4, 8, 24 and 48 h after treatments. Relative expression level of each gene was normalized with 18S rRNA gene. Data are means ± SD of three biological replicates. Asterisks indicate a significant difference (*, P < 0.05; **, P < 0.01, t-test) compared with the corresponding control (0 h).

2015; Zhang et al., 2013). More recently, Huang et al. (2015) reported that members of the OsMC family displayed differential expression patterns in response to abiotic stress. In the present study, all of the HbMCs, except HbMC5, showed transcriptional changes when responding to cold, drought, and salt stresses (Figs. 6e8). Among them, HbMC9 was strongly induced by cold and drought stresses, whereas its expression was not affected by salt stress. However, OsMC7, which is most closely related to HbMC9 in

rice, showed significantly down-regulated expression in leaves after drought, cold, and salt stress treatments. Interestingly, OsMC7, showed opposite expression patterns in root after drought and salt stress treatments (Huang et al., 2015). The expression of HbMC3 was significantly down-regulated by drought, cold, and salt stress. By contrast, HbMC1 was significantly up-regulated by drought, cold, and salt stress (Figs. 6e8). AtMC1, the closest Arabidopsis homologue to HbMC1, was found to be a positive regulator of pathogen-

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triggered PCD (Coll et al., 2014). Therefore, we speculate that HbMC1 may positively regulate abiotic stress-induced PCD. In the present study, HbMC1, HbMC2, HbMC5, and HbMC8 showed significantly higher expression levels in TPD trees than healthy trees, suggesting the involvement of metacaspasemediated PCD in TPD occurrence. Numerous studies have shown that TPD is a complex physiological disorder resulting from over tapping (wounding stress) or over stimulation by ethephon treatment (Faridah et al., 1996; Jacob et al., 1994; Putranto et al., 2015). Interestingly, we found that HbMC1, HbMC5, and HbMC8 displayed up-regulation after wounding treatment (Fig. 9). In addition, they were all up-regulated by ET and MeJA treatments (Fig. 10). In some cases, ET and JA can induce ROS production and trigger PCD (Lam, 2004; Putranto et al., 2015; Zhang and Xing, 2008). Taken together, we speculate that HbMC1, HbMC2, HbMC5, and HbMC8 may be involved in triggering PCD during the onset of TPD syndrome. 5. Conclusion In this study, nine putative metacaspase genes (HbMCs) were identified in rubber tree. Subsequently, their bioinformatic characteristics were systematically analyzed, including conserved domains, sequence identities, exon-intron structures, and phylogenetic relationships. Expression profiling revealed that HbMCs were differently expressed in various tissues and regulated by abiotic stresses, implying that HbMCs may be involved in developmentally and/or environmentally regulated PCD. Additionally, HbMC1, HbMC2, HbMC5, and HbMC8 were found to be associated with TPD. Our results present a comprehensive overview of rubber tree metacaspase gene family and lay an important foundation for further functional characterization of this family in Hevea brasiliensis. Conflict of interest The authors declare that they have no conflict of interest. Author contributions Hui Liu and Dejun Li designed the experiments and wrote the manuscript. Hui Liu, Zhi Deng, Jiangshu Chen, Sen Wang, and Lili Hao performed the experiments, data collection and statistical analysis. All of the authors read and approved the final manuscript. Acknowledgments The research work was supported by the Fundamental Research Funds for Rubber Research Institute, CATAS (1630022014006). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2016.04.011. References Ahmad, R., Zuily-Fodil, Y., Passaquet, C., Bethenod, O., Roche, R., Repellin, A., 2012. Ozone and aging up-regulate type II metacaspase gene expression and global metacaspase activity in the leaves of field-grown maize (Zea mays L.) plants. Chemosphere 87 (7), 789e795. Bollhoner, B., Zhang, B., Stael, S., Denance, N., Overmyer, K., Goffner, D., Van Breusegem, F., Tuominen, H., 2013. Post mortem function of AtMC9 in xylem vessel elements. New Phytol. 200 (2), 498e510. Cabreira, C., Cagliari, A., Bücker-Neto, L., Wiebke-Strohm, B., de Freitas, L.B., Marcelino-Guimar~ aes, F.C., Nepomuceno, A.L., Margis-Pinheiro, M.M., BodaneseZanettini, M.H., 2013. The Lesion Simulating Disease (LSD) gene family as a

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